Extended Data Fig. 4: Related to Fig. 1: The role of SLC36A4 in Trp metabolism in efferocytosing macrophages.
From: Efferocytosis drives a tryptophan metabolism pathway in macrophages to promote tissue resolution
a, Immunofluorescence microscopy of SLC36A4 (green) and LAMP-1 (red) in Scr- or siSlc36a4-transfected BMDMs incubated 45 mins with PKH26-labelled ACs (pseudocolored white); DAPI (blue) nuclear stain. Scale bar, 50 μm. Another set of cells was assayed for Slc36a4 mRNA and immunoblotted for SLC36A4 (n = 3 biological replicates/group). b, Immunofluorescence microscopy of SLC36A4 (green) in BMDMs incubated with PKH26-labelled ACs (red) for 45 min. Scale bar, 50 μm. White arrows, engulfed ACs; blue arrows, unengulfed ACs. c, Representative image of Scr-transfected macrophages not incubated with ACs and then stained for SLC36A4 (green) and DAPI (blue); note low expression of SLC36A4 compared with AC+ macrophages in Fig. 1b. Image is representative of 3 biological triplicates. Scale bar, 50 μm. d, BMDMs were incubated ± apoptotic Jurkat cells (apJCs) or apoptotic macrophages (apMϕs), chased for 3 h, and assayed for Slc36a4 (n = 6 biological replicates/group). e, BMDMs were incubated ± ACs or PS-beads for 1 h, chased for 3 h, and immunoblotted for SLC36A4. f, BMDMs pre-treated with ± 20 µM MG132 were incubated ± ACs for 1 h, chased for 3 h, and immunoblotted for SLC36A4. g, Scr- or siSlc36a4-transfected BMDMs were incubated with PKH26-labelled ACs (red) and quantified for the percentage of PKH26+ macrophages (arrows) of total macrophages. Scale bar, 50 μm (n = 5 biological replicates/group). h, Tryptophan and kynurenine in Scr- or siSlc36a4-transfected BMDMs incubated with ACs (see Fig. 1d; n = 6 biological replicates/group). i, BMDMs were incubated ± apoptotic macrophages for 1 h and assayed for Ido1 (n = 3 biological replicates/group). j, Scr- or siSLC36A4-transfected HMDMs were for SLC36A4 (n = 3 biological replicates/group). All mRNA data are expressed relative to the indicated control groups. Data are mean ± SEM, and significance was determined by two-tailed Student’s t-test or one-way ANOVA with Fisher’s LSD post-hoc analysis.
